Role of intracellular stores in the regulation of rhythmical [Ca2+]i changes in interstitial cells of Cajal from rabbit portal vein

Interstitial cells of Cajal (ICCs) freshly isolated from rabbit portal vein and loaded with the Ca(2+)-sensitive indicator fluo-3 revealed rhythmical Ca(2+)](i) changes occurring at 0.02-0.1 Hz. Each increase in Ca(2+)](i) originated from a discrete central region of the ICC and propagated as a Ca(2+)](i) wave towards the cell periphery, but usually became attenuated before reaching the ends of the cell. In about 40% of ICCs each rhythmical change in Ca(2+)](i) consisted of an initial Ca(2+)](i) increase (phase 1) followed by a faster rise in Ca(2+)](i) (phase 2) and then a decrease in Ca(2+)](i) (phase 3); the frequency correlated with the rate of rise of Ca(2+)](i) during phase 1, but not with the peak amplitude. Rhythmical Ca(2+)](i) changes persisted in nicardipine, but were abolished in Ca(2+)-free solution as well as by SK&F96365, cyclopiazonic acid, thapsigargin, 2-APB, xestospongin C or ryanodine. Intracellular Ca(2+) stores visualised with the low-affinity Ca(2+) indicator fluo-3FF were found to be enriched with ryanodine receptors (RyRs) detected with BODIPY TR-X ryanodine. Rhythmical Ca(2+)](i) changes originated from a perinuclear S/ER element showing the highest RyR density. Immunostaining with anti-TRPC3,6,7 antibodies revealed the expression of these channel proteins in the ICC plasmalemma. This suggests that these rhythmical Ca(2+)](i) changes, a key element of ICC pacemaking activity, result from S/ER Ca(2+) release which is mediated via RyRs and IP(3) receptors and is modulated by the activity of S/ER-Ca(2+)-ATPase and TRP channels but not by L-type Ca(2+) channels.